Our long term objective is to understand how the timing of a developing system is regulated. Virtually all of the basic questions concerning this subject remain partially or completely unanswered. Therefore, we have developed conditional and mutational methods for investigating the number, interrelationships, complexity, and nature of rate-limiting processes, or "timers", in developing systems. These methods will be applied to a single model system, morphogenesis in the slime mold Dictyostelium discoideum. This system exhibits several unusual timing features which make it an exceptional system for such studies. These include: 1) multiple parallel timers, 2) the capacity for rapid recapitulation of the morphogenetic scheme when disaggregated, 3) "erasure, the loss of the capacity for rapid recapitulation, and 4) selective timing mutations. Our specific objectives can be seperated into two portions. First, we will analyze the erasure phenomenon in detail to test whether it represents the selective and complete reversal of the timer pathways. If it does, then identifying what is lost at the time of erasure may present us with clues concerning the nature of timers. So far, erasure appears to be the selective loss of chemotactic responsiveness to cAMP. This investigation will be facilitated by the use of erasure-minus mutants isolated by a new screening procedure described in this proposal. Second, we will continue to refine and develop conditional methods (primarily employing temperature shift experiments), and mutational methods (primarily employing selective timer mutations) for delving into the number, interrelationships, and complexity of timers. By these investigations, we hope to test the hypothesis that "timers" represent a class of pathways which have evolved as fundamental "cues" to regulate developmental programs.